ApoE Peptide-Directed Nanomedicines For Targeted Therapy Of Malignant Glioma

Malignant glioma is characterized by aggressive growth and recurrence,resulting in poor prognosis and a 5-year survival rate<5%.In addition,the blood-brain barrier(BBB)prevents the delivery of most therapeutics to the glioma site.Current clinical treatment is surgical excision,often in combination with chemotherapy(mainly temozolomide)and/or radiotherapy.But the therapeutic effect is usually not ideal,accompanied by side-effects caused by chemodrugs.To deal with these problems,in this thesis,we designed a series of apolioprotein E peptide(ApoE)-functionalized,disulfide-crosslinked nanomedicines for the effective BBB penetration and targeted therapy of glioma with low adverse effect,based on dithiolane trimethylene carbonate(DTC)containing biodegradable copolymers.Chapter 1 reviews the feature,clinical therapy of the glioma and BBB as well as the cutting-edge development of treatment.Chapter 2 firstly reports on the synthetic method of the copolymers for the preparation of glioma-targeting nanomedicines.The ring-opening copolymerization of DTC with trimethylene carbonate(TMC)or ε-caprolactone(CL)initiated by poly(ehthylene glycol)(PEG)and catalyzed by organocatalyst,diphenyl phosphate(DPP)could yield copolymers with well-defined molecular weight and structure,thus facilitating the well-controlled formation process of polymersomes and micelles.Glioma cells have highly active RAF-MEK-ERK(MAPK)signaling pathway that is inactive in normal cells.In chapter 3,we designed sorafenib-loaded,ApoE-directed micelles(ApoE-Ms-SF)self-assembled from biodegradable copolymers PEG-P(DTCCL)-MA and ApoE-PEG-P(DTC-CL)for molecularly targeted therapy of orthotopic U-87 MG-bearing mice.ApoE-Ms-SF with decent SF loading(～9 wt.%)and small size(<30 nm),as compared to non-targeting Ms-SF,could more effectively penetrate BBB and enter U-87 MG cells,leading to stronger cytotoxicity.It could down-regulate pERK and induce apoptosis of U-87 MG cells.In vivo experiment results showed that ApoE-Ms-SF could prolong the blood circulation time of SF and increase the accumulation in glioma,and further significantly delay the growth of U-87 MG tumors and prolong the median survival time of the mice(37 d,**p).Malignant gliomas are highly heterogeneous and prone to mutation,thus the efficacy of ApoE-Ms-SF monotherapy is limited.In chapter 4,we applied ApoE-Ms to co-load an FDA approved photosensitizer indocyanine green(ICG)with SF(ApoEMs-ICG&SF)and combined with laser irradiation for a photo-chemo therapy of orthotopic U-87 MG-bearing mice.The photodynamic therapy(PDT)of photosensitizers produces anti-tumor reactive oxygen species(ROS)in tumor sites,while also results in hypoxic microenvironment and elevated tumor angiogenesis,leading to a rapid proliferation and metastasis of tumors.Whereas SF can suppress angiogenesis and block tumor growth in addition to inhibiting MAPK pathway,the combination of SF and ICG may have synergistic effect.Here ApoE-Ms-ICG&SF with controllable drug loading contents and small sizes(<30 nm)could greatly penetrate BBB and enter U-87 MG cells.By combining with laser irradiation(808 nm),the temperature was increased drastically and vast ROS was generated showing stronger cytotoxicity to U-87 MG cells.In vivo experiment results demonstrated that ApoE-MsICG&SF plus laser irradiation significantly suppressed the tumor development and prolonged the median survival time of mice(48 d).Immunotherapy is a promising alternative to prevention of tumor progression to relief of tumor relapse after traditional chemotherapy.Immunotherapy is a hopeful way to delay tumor progression for a long time to relieve the tumor relapse after traditional anti-tumor drugs discontinuation.However,malignant gliomas have an immunesuppressive tumor microenvironment and BBB prevents immunostimulatory drugs entering the glioma site,resulting in<10%response.In chapter 5,we utilized chimaeric polymersomes based on PEG-P(TMC-DTC)-sp and ApoE-PEG-P(TMC-DTC)to load CpG oligodeoxynucleotide(CpG),a toll-like receptor 9(TLR9)agonist(ApoE-PSCpG),and investigated the targted immunotherapy of malignant orthotopic murine glioma LCPN-bearing mice.Here ApoE-PS-CpG with small and uniform sizes(～40 nm)and high drug loading(10-20 wt.%)could efficiently penetrate BBB,target to glioma sites and cervical lymph nodes,thus strongly stimulate the maturation of antigen-presenting cells(APCs)and the production of pro-inflammatory cytokines in vitro and in vivo.Notably,both intravenous or intranasal administration of ApoE-PSCpG significantly prolonged the median survival time of orthotopic LCPN-bearing mice,and the combination with radiotherapy further enhanced the immunotherapeutic effect.This intelligent ApoE-PS-CpG nano-immunoadjuvant could realize the BBB penetation and the protection from degradation of CpG,and circumvent the invasiveness and side-effects related with the intracranial or intrathecal injection for glioma therapy,thus broadens the therapeutic application of CpG as a universal nanoimmunoadjuvant for the immunotherapy of glioma.The delivery of tumor antigen-generating drugs like granzyme B(GrB)to glioma cells may further enhance the efficacy of immunotherapy of ApoE-PS-CpG.In chapter 6,we designed GrB-loaded,ApoE-directed polymersomes(ApoE-PS-GrB)and applied it in combination with ApoE-PS-CpG for enhanced immunotherapy of orthotopic LCPN-bearing mice.ApoE-PS-GrB could significantly penetrate BBB,and enter LCPN cells,followed by effectively escape from the endo/lysosomes and the damage of the mitochondrial membrane.This eventually led to immunogenic cell death(ICD)of LCPN cells and the release of tumor antigens,inducing strong cytotoxicity and favorable immune microenviroment.Remarkably,in the immunotherapy of orthotopic LCPN mice,the systemic delivery of combo of ApoE-PS-GrB and ApoE-PS-CpG could induce ICD of LCPN cells,and compared to the monotherapy,could significantly enhance the activation of dendritic cells(DCs),recruitment of cytotoxic lymphocytes(CTLs)and secretion of pro-inflammatory cytokines,while decrease the inhibitory T cells and M2-type macrophages in tumor microenvironment.The treatment with the combo showed superior tumor inhibition and the survival prolonging of the mice to the monotherapy.Therefore,the combo of ApoE-PS-GrB and ApoE-PS-CpG via intravenous injection could effectively reduce/eliminate side-effects associated with intracranial or intrathecal injection,and increase tumor antigens and reverse the immunosuppressive microenvironment in gliomas.Chapter 7 summarizes the whole thesis,discusses the issues associated with the thesis,and gives perspecitves on future development of effective glioma therapy.